Project description:Pure cultures of N. viennensis were subjected to copper limitation using the copper chelator TETA. Two samples that were subjected to copper limiation for approximately two weeks were used for protein extraction and subsequent proteomic analysis.
Project description:Ammonia-oxidizing archaea (AOA) are among the most abundant microorganisms and key players in the global nitrogen and carbon cycles. They share a common energy metabolism but represent a heterogeneous group with respect to their environmental distri- bution and adaptions, growth requirements, and genome contents. We report here the genome and proteome of Nitrososphaera viennensis EN76, the type species of the archaeal class Nitrososphaeria of the phylum Thaumarchaeota encompassing all known AOA. N. viennensis is a soil organism with a 2.52-Mb genome and 3,123 predicted protein-coding genes. Proteomic analysis revealed that nearly 50% of the predicted genes were translated under standard laboratory growth conditions. Comparison with genomes of closely related species of the predominantly terrestrial Nitrososphaerales as well as the more streamlined marine Nitrosopumilales (Candidatus order) and the acidophile Nitrosotalea devanaterra revealed a core genome of AOA comprising 860 genes, which allowed for the reconstruction of central metabolic pathways common to all known AOA and expressed in the N. viennensis and Nitrosopelagicus brevis proteomes. Concomitantly, we were able to identify candidate proteins for as yet unidentified crucial steps in central metabolisms. In addition to unraveling aspects of core AOA metabolism, we identified specific metabolic innovations associated with the Nitrososphaerales mediating growth and survival in the soil milieu, including the capacity for biofilm formation, cell surface modifications and cell adhesion, and carbohydrate conversions as well as detoxification of aromatic compounds and drugs.
Project description:A band containing the ammonia monooxygenase complex from Nitrosospheara viennensis was cut from a Blue Native PAGE gel and cross linked using DSSO.
Project description:Information on the files related to the respective experiments can be found in the metadata file titled “Metadata_for_Nviennensis_Experiments.xlsx”. The data found here encompasses three related mass spectrometry experiments: 1.) BN-PAGE: Biomass of Nitrososphaera viennensis grown in a bioreactor was lysed and membrane fractions were harvested using an ultracentrifuge. Membrane proteins were extracted using n-dodecyl-beta-D-maltoside (DDM) and loaded on a 3-12% gradient blue native PAGE gel. Selected bands were cut out and analyzed via mass spectrometry. 2.) SDS-Tricine-PAGE of AMO Band: A band containing a large amount of the ammonia monooxygenase complex (AMO) was denatured and run on a 15% SDS-Tricine-PAGE gel to identify individual subunits. 3.) AmoC from SDS-Tricine-PAGE: A band containing a high amount of AmoC from the SDS-Tricine-PAGE gel was processed with three separate digestive enzymes (trypsin, chymotrypsin, or GluC) to identify unique peptides among the six AmoC homologs found in the genome of N. viennensis.
